Isolation, taxonomic analysis, and phenotypic characterization of bacterial endophytes present in alfalfa (Medicago sativa) seeds.

A growing body of evidence has reinforced the central role of microbiomes in the life of sound multicellular eukaryotes, thus more properly described as true holobionts. Though soil was considered a main source of plant microbiomes, seeds have been shown to be endophytically colonized by microorganisms thus representing natural carriers of a selected microbial inoculum to the young seedlings. In this work we have investigated the type of culturable endophytic bacteria that are carried within surface-sterilized alfalfa seeds. MALDI-TOF analysis revealed the presence of bacteria that belonged to 40 separate genera, distributed within four taxa (Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes). Nonsymbiotic members of the Rhizobiaceae family were also found. The evaluation of nine different in-vitro biochemical activities demonstrated isolates with complex combinations of traits that, upon a Principal-Component-Analysis, could be classified into four phenotypic groups. That isolates from nearly half of the genera identified had been able to colonize alfalfa plants grown under axenic conditions was remarkable. Further analyses should be addressed to investigating the colonization mechanisms of the alfalfa seeds, the evolutionary significance of the alfalfa-seed endophytes, and also how after germination the seed microbiome competes with spermospheric and rhizospheric soil bacteria to colonize newly emerging seedlings.

Specificity traits consistent with legume-rhizobia coevolution displayed by Ensifer meliloti rhizosphere colonization.

Rhizobia are α- and ß-proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we performed a genome-wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet-unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe.

Novel tnpR-based transposable promoter traps suitable for RIVET studies in different gram-negative bacteria.

The preparation of plasmid-borne RIVET libraries can be troublesome when high genomic coverages are needed. We present here the construction and functional validation of a new set of miniTn5 promoter traps to generate tnpR-based RIVET libraries. The ability to generate tnpR transcriptional fusions by transposition will significantly facilitate the setup of RIVET studies in those bacteria where Tn5 transposition is operative.

Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria.

Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own helper functions. The results present an as-yet-unclassified and seemingly ubiquitous conjugal system that provides a mechanistic support for the HGT between sympatric rhizobia of Medicago roots, and between other soil and rhizospheric bacteria.

Prevalencia de fluorosis dental y anomalías de desarrollo del esmalte en los niños de la Escuela Bolivariana "Juan Ruiz Fajardo" del Estado Mérida, Venezuela / Prevalence of dental fluorosis anomalie enamel developmente in children Escuela Bolivariana \"Juan Ruiz Fajardo\" Estado Mérida, Venezuela

Determinar la frecuencia de Fluorosis Dental, Opacidades e Hipoplasias del Esmalte, en niños en edad escolar entre 5 y 14 años, de la Escuela Bolivariana "Juan Ruiz Fajardo", del Estado Mérida, Venezuela. Se realizó un estudio descriptivo transversal. La población estuvo constituida por 445 niños entre 5 y 14 años de edad, de la Escuela Bolivariana "Juan Ruiz Fajardo" del Estado Mérida, Venezuela. Se estudiaron las variables fluorosis dental, opacidades e hipoplasia del esmalte, aplicando el Índice de Dean y el Índice de Desarrollo de Defectos del Esmalte (DDE) modificado, según metodología descrita por la Organización Mundial de la Salud (OMS). El 80,7% de la población estudiada no presentó fluorosis dental, en contraste con el 19,3% que si presentó, a predominio de fluorosis dudosa en un 9,9% y fluorosis muy leve en un 6,3%. El 76,2% de la población no presentó alteraciones en el desarrollo del esmalte, en contraste con un 12,4% y 3,6% que presentó opacidades delimitadas y difusas respectivamente. Se evidencia que el mayor porcentaje de la población estudiada no presenta fluorosis dental ni alteraciones en el desarrollo del esmalte. Sólo un pequeño porcentaje presentó fluorosis dudosa y muy leve, además de opacidades delimitadas y difusas

To determine the frequency of dental fluorosis, enamel opacities and hypoplasia in 5-14-year-old-school children from the Bolivariana School "Juan Ruiz Fajardo" in Mérida, Venezuela. The study was descriptive and transversal. Participants were 445 children aged between 5 y 14 years. Children were attending the Bolivariana School "Juan Ruiz Fajardo" in Mérida, Venezuela. The variables dental fluorosis, enamel opacities and hypoplasia were considered by using Dean's Fluorosis Index and the Enamel Defect Index (EDI) Modified, according to WHO's methodology. 80.7% of the studied population did not show signs of dental fluorosis, opposite to 19.3% that presented fluorosis, with a higher score for doubtful fluorosis equal to 9.9%. In relation to alterations in enamel 76.2% of the participants did not present alterations in contrast to 12.4% and 3.6% who presented delimitated and diffuse opacities, respectively. It was evident that the higher percentage of the participants did not show either dental fluorosis or alterations in enamel development. Just a small percentage of children presented doubtful and very mild fluorosis as well as both delimited and diffuse opacities

First genomic analysis of the broad-host-range Rhizobium sp. LPU83 strain, a member of the low-genetic diversity Oregon-like Rhizobium sp. group.

Alfalfa (Medicago sativa) is the most cultivated forage legume for cattle and animal feeding, occupying about 32 million hectares over the world. Management of the N2-fixing symbiosis of this plant to maximize crop production is therefore an important objective. A fundamental constraint to this aim emerges when a moderately low soil pH hampers the establishment of an effective symbiosis with indigenous and/or inoculated rhizobia. Besides the association of alfalfa with Ensifer (Sinorhizobium) meliloti, this legume is able to establish a symbiosis with Ensifer (Sinorhizobium) medicae and with less characterized types of rhizobia, such as the Oregon-like strains, Rhizobium sp. Or191 initially isolated in the USA, and the Rhizobium sp. LPU83 strain, from Argentina. These strains are acid-tolerant, highly competitive for acidic-soil-alfalfa nodulation, but inefficient for biological nitrogen fixation with alfalfa. These features position the Oregon-like rhizobia as strains of potential risk in agricultural soils compared with the efficient symbiont E. meliloti. Moreover, the collected genetic information has revealed that the genomic structure of these rhizobial isolates is complex in terms of sequence similarities shared with other rhizobia. Such a "patched" genetic composition has obviously imposed severe restrictions to the classical taxonomy of these rhizobia. In this work we summarize the accumulated knowledge about the Oregon-like rhizobia and present a phylogenetic analysis based on genome sequence data of Rhizobium sp. LPU83 obtained by a high-throughput sequencing on the Genome Sequencer FLX Titanium platform. The accessibility of the complete genomic sequence will release up more experimental possibilities since this information will then enable biochemical studies as well as proteomics and transcriptomics approaches.

Traumatismo oro-facial por mordedura canina: presentación de un caso clínico / Oro facial trauma caused by dog bites: a case report

Los traumatismos provocados por mordidas caninas o por otros animales domésticos suelen traer un mosaico de lesiones y secuelas con signos y síntomas variados. Cada caso es de por sí una entidad diferente que amerita manejo interdisciplinario. El objetivo, destacar la importancia del manejo interdisciplinario de pacientes pediátricos con traumatismos oro-faciales y reportar un caso clínico con mordedura canina. Revisión bibliográfica de la epidemiología, diagnósticos y terapéuticas. Se trata de un paciente preescolar femenino, 3 años de edad, mordida por el perro de su casa, atendida multidisciplinariamente en el Hospital Universitario de Maracaibo por presentar lesiones en región cráneo-facial, acompañadas de hemorragia intra-oral por laceraciones de labios y mucosa, pérdida de hueso alveolar, avulsión y subluxación dentaria además de lesiones en extremidades superiores. Se presenta seguimiento y resolución del caso. El manejo interdisciplinario del paciente con traumatismo oro-facial asegura una atención de emergencia oportuna, restauradora y de mejor pronóstico.